The carbohydrate-binding module of TrCel7A aids in navigating hemicellulose barriers in plant cell walls

Efficient enzymatic deconstruction of plant cell walls is critical for the utilization of lignocellulose biomass. Key enzymes in this process are cellobiohydrolases, a class of cellulases that processively degrade crystalline cellulose. Many cellobiohydrolases possess a carbohydrate-binding module (CBM), yet the specific roles of CBMs in substrate interaction remains unclear. Here, we use single-molecule fluorescence microscopy to investigate how CBM1 of Trichoderma reesei Cel7A influences enzyme binding and motility on cellulose substrates of varying complexity. We compare WT Cel7A with a truncated variant lacking CBM1 (Cel7A^ΔCBM) on bacterial cellulose, phosphoric acid swollen cellulose, delignified milkweed cellulose, and holocellulose nanofibrils (hCNFs). While both variants showed similar steady-state binding densities on bacterial cellulose and phosphoric acid swollen cellulose, Cel7A^ΔCBM exhibited reduced binding on milkweed cellulose and hCNF, with the greatest reduction on the hemicellulose-rich hCNF. Alkali treatment of hCNF to remove hemicellulose partially restored Cel7A^ΔCBM binding, suggesting a role for CBM1 in substrate navigation and productive binding site recognition. Kinetic analyses revealed that CBM1 enables a rapid binding mode absent in the truncated variant. Consistent with this, the isolated CBM1 domain had a faster substrate association rate than the isolated catalytic domain. These findings demonstrate that CBM1 enhances cellulase-substrate interactions by accelerating binding and enabling navigation of the complex environment of plant cell walls. Our results emphasize the importance of CBMs in natural cellobiohydrolase function and highlight their value in the design of improved cellulases for industrial biomass conversion.